Viruses manipulate cellular processes to create conditions conducive to their own replication. Since viruses possess small genomes that do not encode all the factors required for DNA replication, they rely on host cell proteins to propagate their genomes. Active recruitment of cellular proteins to viral replication compartments functions to promote virus gene expression and replication. In contrast, viral early proteins target antivira cellular DNA sensors and transcriptional repressors to prevent their access to viral genomes. Studying viral replication and virus-host interactions has been hampered by the lack of technologies to identify cellular proteins involved in viral DNA replication. Our contribution in tis proposal will be the application of a novel technology to identify viral and cellular proteins that associate with replicating viral genomes. Our central hypothesis is that cellular DNA replication/repair proteins are recruited to viral genomes to function with viral- encoded replication factors, and that early viral proteins prevent access to viral genomes by antiviral factors with detrimental outcomes for viral replication. We propose to employ a recently described technology that identifies proteins on replicating DNA by coupling Isolation of Proteins on Nascent DNA (iPOND) with Mass Spectrometry (MS). We will use iPOND-MS to identify proteins that interact with viral DNA during infection. To test the feasibility of this innovative technology we have chosen herpes simplex virus type 1 (HSV-1). Herpesviruses are ideally suited to this approach because they block host cell DNA replication, and generate large numbers of viral genomes during lytic replication. HSV-1 encodes the immediate early protein ICP0 that promotes viral gene expression and also overcomes intrinsic host defenses. Guided by strong preliminary data, we will test our hypothesis by pursuing two Specific Aims that will (i) identify cellular proteins specifically exploited by HSV-1 viral DNA replication and (ii) determine how viral ICP0 alters the spectrum of proteins associated with HSV genomes. Comparing proteins identified on replicating viral genomes to the inventory of proteins on active cellular replication forks will reveal differences between viral and cellular DNA replication. Proteins recruited specifically by viruses could represent potential targets for antivirals that would block virus propagation. Comparing proteins associated with the genomes of wild-type HSV-1 and ICP0 mutants will reveal how ICP0 recruits factors to promote gene expression, while also manipulating cellular responses to prevent recognition and inhibition on viral genomes. Our approach is innovative because it represents a completely new large-scale approach to identify proteins that are recruited to aid viral transcription and replication. We anticipate this new technology will be broadly applicable to many other DNA viruses. Identifying cellular proteins commonly exploited for viral DNA replication across different virus families will suggest potential targets for development of novel broadly acting antiviral therapeutics.